J. Mol. B601. (1975) 96, 291-306

Identification of Discrete Electrophoretic Components among the Products of Mitochondrial Protein Synthesis in HeLa Cells PAOLO COSTANTINO ANI) GIVSEPPE ATTARDI Division

(Rereived

of Biobgy, California Imtitute of Technology Pa.scdena, Ccblif. 91125, C7.S.A. 6 August

1974, and iti revised

form

11 April

1.975)

Proteins synthesized in wiwo by HeLa cell mitochondria, in the presence of emetine, an inhibitor of cytoplaamio protein synthesis, have been characterized as to their electrophoretic mobility, solubility properties in organic solvents, and kinetics of labeling with [3H]isoleucine. Ten distinct electrophoretic components in the molecular weight range from about 11,000 to 42,000 have been identified with high reproducibility among the products of mitochondrial protein synthesis. Control experiment8 involving direct sodium dodecyl sulfate lysis of whole cells, or the use of the protease inhibitor phenylmethylsulfonylfluoride during cell homogenization and fractionation, have excluded a significant role of enzymic degradation during extraction in producing the observed electrophoretic pattern of mitochondrial protein products. A selective solubilization of mitochondrial protein products, representing a 20 t.o SO-fold purification with respect to the cytoplasmically synthesized proteins, has been obtained by using a neutral chloroform/methanol mixture. At least six of the electrophoretic components with a molecular weight greater than 20,000 were found to be extracted to an appreciable extent with neutral chloroform/methanol. A fairly co-ordinate labeling of the discrete electrophoretic components has been observed during a one-hour exposure period of the cells to C3H]isoleucine in t Ifa presence of ometine, u-ithout any clear evidnrtcc: of intcrconvtwior~.

1. Introduction Relatively

simple

electrophoretic

profiles

have

beeu

reported

prel;iously

by

several

of HeLa cells (Galper 8t Darnell, 1971; Brega & Baglioni, 1971; Lederman & Attardi, 1973), hamster BHK 21 cells (Coote 8t Work, 1971), mouse L cells (Kiehn & Holland, 1970), yeast (Thomas & Williamson, 1971; Weislogel & Butou, 1971; Tzagoloff & Akai, 1972; Schatz et al., 1972; Ebner et al., 1973), Neuroqoro craSsa (Sebald et al., 1971). and i?a vitro by rat liver and hamster liver mitochondria (Coote & Work, 1971; Bosmann. 1971; Burke & Beattie, 1973). Although the number of distinct polypeptides synthesized in mitochondria is presumably small (Beattie, 1971), in only a few of t,he studies mentioned above, most on yeast mitochondria, was the resolution and reproducibility of the patterns observed sufficient to allow the identification and molecular weight estimation of some of the individual protein components (Thomas Rr; Williamson, 1971; Weislogel $ Butow, 1971; Tzagoloff t Akai, 1972: Schatz et ccl.. 1972; Ebner et al. 1973; Burke & Beattie, 193). authors

for

the

proteins

synthesized

in

viva

by

mitochondria

292

I’.

COSTAh;‘l’lNO

ANO

U.

A’l”l’hlC~l

In the present work, improvements in the preparation of the sample and in the conditions of elect,rophoresis have allowed the identification, among 6hc itb vilto mitochondrial products in HeLa cells, of ten distinct rlectrophoret~ic componrms in the molecular weight range from about 11,000 to 42,000. Thcsc componcms hart been analyzed as to their solubility in organic solvents and kinetics of labeling. A substantial purification of at least six of these components has been obt,aincd based on their selective solubility in a neutral chloroform/methanol mixt’urc.

2. Materials

and Methods

(a) Growth Suspension The cultures

cultures

were

free

of HeLa cells ~vere grown of detectable Myco&s?rha (b) Labeling

of cells as described conta,mination.

by Aiualdi

& Attarcli

(1968).

conditiovla

Por pulse-labeling of the products of mitochondrial prot,rin synthesis, cells \vere ~vashnd twice with isoleucine-free modified Eagle’s medium (Levintow & Darnell, 1960) containing 5% dialyzed calf serum, and resuspended in the same medium at a concont~ration of 1.0 x lo6 to 1.5 x lo6 cells/ml, unless otherwise specified. After 30 min of adaptation, tlir: cells were treated for 5 min with 100 pg ometine/ml, tt specific inhibitor of cytoplasmic protein synthesis (Perlman & Penman, 1970; Ojala & Attardi, 1972), or 100 pg cmetine/ml and 100 pg chloramphenicol/ml, an effective inhibitor of mitochondrial protein synt,hcsis (Kroon, 1965; Linnane, 1968; Lederman & Attardi, 1970), and then exposed, iii thr, presence of the antibiotic(s), to L-[4,5-3H]isoleucine (30 to 50 Ci/mmol; 7 to 15 &i/ml, unless otherwise specified) for various lengths of time, as described in the legends to the Figures. For the analysis of the products of mitochondrial protein synt,hesis in whole-cell small-scale experiments were carried out using 5-ml sodium dodecyl sulfate lysates, suspension cultures at 2.5 x lo6 cells/ml and n-[4,5-3H]isoleucine at 100 &i/ml. In some experiments, the labeling was performed in the presence of 1 pg ethidium bromide/ml or 100 pg puromycin/ml, added at the same time as emetine. Long-term labeling of total HeLa cell proteins was carried out by growing the cells foi 48 to 72 h in modified Eagle’s medium containing lysine and arginine at 2 x 10s4 M each, with the addition of L-[14C]lysine (318 Ci/mol; 0.0063 to 0.063 &i/ml) and L-[14C]arginine (318 Ci/mol; 0.0063 to 0.063 &i/ml). (c) i%bcellidar The 5000 g,, mitochondrid supernatant of the cytoplesmic In some experiments, the at a concentration of 1.0 inM mitochondrial fraction. For the analysis of whole-cell Tris buffer (pH 7.0 at 25”C), sonicated for 10 s (twice for dialyzed for 18 h against 0.5%

fractionation

fraction (Loderman & Rttardi, 1970) ad the 13,300 g,, extract were prepared as described by Attardi et al. (1969). protaase inhibitor phenylmetl~ylsulfonylfluoride was added to the cell homogenate and to the media used to wash the lysates, the labeled cells were lysed with 3.0 ml 0.01 M0.1 M-NaCl, 0.001 M-EDTA, 0.5% sodium dodecyl sulfate, 5 s, at room temp.) with a Branson sonifier (see below) and sodium dodecyl sulfate (150 vol., 3 changes).

(d) Disruption of the mitochondriul membranes fraction was resuspended in 0.25 M-SUCroSe, 0.01 5000 g,, mitochondrial acetate buffer (pH 7.0), at a concentration of 2 to 4 mg protein/ml, as determined Lowry method. The suspension was, in most cases, sonicated in a volume of 1 for 20 s (twice for 10 s in ice-water) with a Branson sonifier (model S-125) 7 A, using a microprobe. In some cases, the sonicate was centrifuged at 30,000 20 min and the resulting supernatant recentrifuged at 105,000 g,, for 3 h. In some ments, the mitochondrial membranes were disrupted by treatment with 0.2% Triton 3100, or 0.296 sodium deoxycholato or digitonin at 100 pg/mg protein. The

nz-Tris by the to 3 ml set at gaV for experior 2%

MITOCHONDRIAL

PROTEIN

(e) Chloroform/methanol

SYNTHESIS

293

extraction

Samples of the sonically disrupted 5000 g,, mitochondrial fraction (0.2 to O-7 mg protein in 0.05 to 0.15 ml) or of the pellets obtained by high or low speed centrifugation of the sonicate, as described above, were mixed with 2 ml of a mixture 2: 1 (v/v) of chloroform and methanol (neutral extraction). In some experiments, the chloroform/methanol mixture was acidified with 2 x 10V3 M-HCl (acid extraction). The suspensions were then incubated cGt,her at 50°C for 30 min, or at 70% for 45 min, and the insoluble residues pelleted b) centrifugation at 1500 g,, for 5 min. The extracts and the residues were dried under llit,rogen and dissolved in 0.2 ml 2.5?$ sodium dodecyl sulfate, which n-aa then neutralized in the case of the acid extraction. In some cases, the samples were treated with 907; methanol at room temperature for IO min, and the precipitated material was separated by centrifugation and &en extracted with chloroform/methanol. (f)

Polyacrylamide

gel electrophoresis

Tllo system described by Weber & Osborn (1969) was used, with the modification that acrylamide cross-linked with 0.313% N,N’-methylene-bisacrylamide was used. 12.50,; Electrophoresis was carried out through 0.6 cm x 23 cm gels at 7.5 mA/gel in a Canalco model 1400 apparatus for 13 to 43 h, as indicated in the Figure legends, at room temperature. The gels were fractionated aa described previously (Lederman & Attardi, 1973). All samples to be applied to the gels, dissolved in 0.2 ml 2.5% sodium dodecyl sulfate, were brought to 5% mercaptoethanol and, in most cases, heated at 70°C for 30 min. In some experiments, before the heating step, the sodium dodecyl sulfate lysates were treated for 30 min at pH 10, 37”C, to break any peptidyl-tRNA complexes, and then neutralized. In other experiments, the lysates were brought to 2 x lob2 M-NaOH, heated at 70°C and electrophoresed without neutralization. Parallel gels of marker proteins, treated as the radioactive samples, were run routinely. The following proteins were used in various combinations in these runs and in the calibration experiment shown in Fig. 2(f) : bovine serum albumin (M, = 66,000); y-globulin heavy chain (M, = 50,000); ovalbumin (M, = 43,000) ; pepsin (M, = 35,000); chymotrypsinogen A (M, = 25,700); y-globulin light chain (M, = 23,500); trypsin (M, = 23,300) ; myoglobulin (M, = 17,200) ; lysozyme (M, .;: 14,300); ribonuclea,se A (M, = 13,700).

3. Results (it) Electrophoretic

analysis

of the products

of mitochondrid

protein

synthesis

work, the membranes of the 5000 g,, crude mitochondrial cells were in most ca,ses disrupted by extensive sonica,tion: t,he resulting suspension, after addition of 2.50,; sodium dodecyl sulfate and 59>, mercaptoethanol, were heated at 70°C for 30 min. In more recent experiments, the heating step was omitted without noticea,ble effects. Figure 1 shows the kinetics of accumulation of radioactivity in the proteins of fraction during exposure of HeLa cells to [3H]isoleucine t’he 5000 g,, mitochondrial for one hour in the presence of 100 pg emetine/ml. It has been shown that the great majority of the proteins synthesized in HeLa cells under these conditions represent products of mitochondrial protein synthesis (Perlman 8: Penman, 1970; Ojala $ Mardi, 1972; England & Attardi, 1974).t It appears that the labeling of t,hcsc In

the

fract,ion

present

from

HeLa

in these cells has been found t In particular, more than 99:; of total amino acid incorporation to be inhibited by concentrations of emetine greater than 4 pg/ml (Perlmen & Penman, 1970). Of the amino acid incorporation that is resistant to 100 &ml of the drug bbout 70% appears to be sensitive to chloramphenicol (England & Attardi, 1974); furthen$ore, the distribution of the emetine-resistant, chloramphenicol-resistant incorporation among different centrifugal fractions suggests that it is associated with rough endoplasmic reticulum elements (England & Attardi, 1974).

294

P. COSTANTlNO

AND

G. ATTARDl

Tlmc (mm)

Pla. 1. Kinetics of accumulat~ion of radioactivity in the products of mitochondrial protein synthesis in HeLa cells. HeLa cell oultures ( lo6 cells/ml) were labeled with [3H]isoleucine (7 &i/ml) for various lengths of time in the presence of emetine, aa described in Materials and Methods, section (b). The mitoohondrial fraotion was isolated from eaoh sample and sonicated, aa described in Materials and Methods, sections (a) and (d), and the trichloroacetic acid-precipitable radioactivity and the protein ooncentration were measured in samples of each fraction.

proteins continues at a decreasing rate for at least one hour. This decrease is not due to exhaustion of the isotope in the medium. Figure 2 shows the profiles obtained by running the sodium dodecyl sulfate lysates of the sonicated mitochondrial fraction from cells labeled with [3H]isoleucine through 12.5% polyacrylamide gels for 26 hours for different times. After a three-minute [3H]isoleucine pulse (Fig. Z(a)), there appears to be a rather uniform distribution of radioactivity in the region corresponding to the molecular weight range from 10,000 to 50,000, with a fairly prominent broad peak centered around 9000. Addition of chloramphenicol at 100 pg/ml, at the same time as emetine, suppresses almost completely the labeling of the material in the 10,000 to 50,000 molecular weight range, without affecting the labeling of the broad peak around 9000. After a ten-minute labeling (Fig. 2(b)), the electrophoretic profile of the products of mitochondrial protein synthesis shows several distinct peaks of radioactivity in the region between M, = 20,000 and 45,000, and a smaller one at about M, = 11,000, emerging over a high background of heterogeneous material ; the peak around 9000 daltons, on the contrary, is relatively less pronounced, and there are now some chloramphenicol-sensitive heterogeneous products migrating to that region of the gel. After one hour labeling (Fig. 2(c)), the electrophoretic profile is very similar to that after ten minutes labeling, except that there is no clearly recognizable 11,000 component and no evidence whatsoever of the broad 9000 peak. In a shorter electrophoretic run (13 h) of a 30-minute labeled sample, no discrete labeled component migrating faster than material with M,. = 9000 was observed. In order to investigate the possible contribution of degradative phenomena to the observed electrophoretic profiles of the products of mitochondrial protein synthesis, cells labeled for one hour with [3H]isoleucine in the presence of emetine were harvested, washed and immediately lysed with sadium dodecyl sulfate; the lysate was then sonicated, dialyzed, and run on a gel. The ljrofile obtained (Wig. 2(d))

MITOCHONDRIAL

BSA I

2.0

OA i

PEP i

RNAase 1

PROTEIN

(0

I~, ’

SYNTHESIS

BSA

201

OA PEP

,,

RNAase 1

(b)

40, I I 2.01

Miyra!lon

(cm)

FIG. 2. Electrophoretic profiles of the products of mitochondrial protein synthesis from HeLa cells labeled for various lengths of time with [sH]isoleucine in the presence of emetine (26 h runs). Samples containing 100 to 200 pg protein were applied to 12.6% polyacrylamide gels and electrophoresed for 26 h, as described in Materials and Methods, se&ion (f). (a), (b) and (c) Lysates of the mitochondrial fraction from cells labeled with [3H]isoleucine for 3, 10 and 60 min, respectively, in the absence (--O-e--) or presence (-- 0 -- O--) of chloramphenicol. (b) The profile obtained with an equal sample of the IO-min labeled mitochondrial fraction, which had been repeatedly frozen and thawed, is also shown (-- A -- A --) (only onethird of each fraction of the gel ww counted in this case). (d) Sodium dodeoyl sulfate lysate of whole cells ( -lOs), labeled for 60 min. from cells labeled for 3 min in the presence of emetine and chlor(e) 13,300 g., supernatant amphenicol. (f) Calibration of the polyacrylamide gels with standard proteins (20 pg of each) run for 26 h under the same oonditions as the radioactive samples. Some of the markers do not appear to obey the expected linear relationship between logarithm of molecular weight and migration. Similar deviations, leading to lower apparent moleaular weights for bovine serum albumin and lysozyme, have been reported by Dunker & Rueckert (1969). The numbers on the peaks oorrespond to those shown in the profiles of the longer electrophoret,ic runs (Fig. 3). BSA, bovine serum albumin; OA, ovalbumin; PEP, pepsin; y-GH, y-globulin heavy chain; y-GL, y-globulin light chain; CT, chymotrypsinogen A; TRY, trypsin; MYO, myoglobin; LYS, lysoeyme; RNAase, ribonucleaso .4.

296

P.

COSTANTINO

AND

G.

ATTARDI

was very similar to that observed after isolation of the mitochondrial fraction (Fig. 2(c)), as concerns both presence and relative amount of the discrete components; this result argues against any significant role of proteases during the isolation and handling of the mitochondrial fraction. Additional evidence in the same direction will be presented below (see Fig. 5(a)). A progressive decrease in the amount of the material in the peak with 31, = 9000 was observed on repeated freezing and thawing of the sonicated mitochondrial -1 (a)

;

BSA t

OA I

(4

i I

BSA

OA PEP

Migration

i

1,

PEP t

(b)

Cd)

(cm)

FIG. 3. Electrophoretic profiles of the products of mitoohondrial protein synthesis from HeLrt cells labeled for different lengths of time with [sH]isoleucine in the presence of emetine (40 h runs). (a), (b), (c) and (d) The mitochondrial fraction was isolated from cells labeled with [3H]isoleucine for 10, 20, 30 and 60 min, respectively, as described in Materials and Methods, section (b). After soniamtion, semples containing 100 to 200 pg protein, dissolved in 2.6% sodium dodecyl sulfate, or 2.6% sodium dodecyl sulfate, 6% 2 x 10-s M-NaOH, 6% mercaptoethanol (-a-@--), meroaptoethanol (-- 0 -- 0 --), were applied onto 126o!0 polyacrylamide gels, and electrophoresed for 40 h, as described in Materials and Methods, section (f). (e) snd (f) A high specific activity prepsration was made by labeling HeLa cells for 1 h with 100 PCi [3H]isoleucine/ml in the presence of emetine. The insert of (f) shows the electrophoretjic profile of the mitochondriel lysate in a 43 h run. The material in the peak fraction of each one of components 1 to 8 was lyophilized and reele&rophoresed for 24 h ((e) and (f)). See the legend to Fig. 2 for abbreviations used.

MITOCHONDRIAL

PROTEIN

SYNTHESIS

207

fraction (see e.g., Fig. 2(b)). However, the labeling of this material, during a fiveminute [3H]isoleucine pulse, was found to be insensitive to both ethidium bromide and puromycin and, therefore, its nature was not investigated further. In order to obtain a better resolution of the products of mitochondrial protein runs synthesis and a more reliable estimate of their size, longer electrophoretic (40 h) were carried out on material from cells labeled for 10 to 60 minutes wit)h [3H]isoleucine in the presence of emetine. Under these conditions, other discrete electrophoretic components, beside those seen in the 26-hour runs, can be identified in the radioactivity profiles. In particular, eight distinct peaks, designated here with the numbers 1 to 8, are more or less clearly recognizable after all labeling times. Most prominent are components 3, 5 and 8. In some prepa,rations, a ninth component) is recognizable reproducibly in repeated electrophoretic runs (Fig. 3(a) and (I,): see also Fig. 2(d)), while in others it cannot be identified against the high heterogeneous background. Component 10, migrating faster than RNAase, which could be seen in the shorter runs (Fig. 2(b)), h as, under t’he present conditions, migrated out of the gels. The electrophoretic profiles of the products of mitochondrial protein synthesis from cells labeled for different times (Figs 2 and 3) do not show substantial differences in the relative labeling of the discrete components. However, there is a progressive decrease in the proportion of radioactivity associated with the heterogeneous material that underlies the distinct peaks. Furthermore, the labeling of component 5 shows :I marked and reproducible decline, relative to that of the other components, in t,he one-hour pulse. Unresolved components moving slower than component 1, and sometimes overlapping it, could be seen in variable amounts in different gel runs. with a tendency to become relatively more abundant after longer labeling times. (See e.g. insert in Fig. 3(f), where they form a broad peak on the slower moving side of component 1.) It should be noticed that the electrophoretic patterns of t’he products of mitochondrial protein synthesis showed a considerable degree of reproducibility, and profiles similar to those shown in Figures 2 and 3, with all discrete components recognizable, were obtained in a variety of experiments involving different preparations and va,rging conditions of treatment of the material. Jn particular, disruption of the mitochondrial membranes by exposure to O-2 or 2% Triton X100, or O.Sq;, sodium deoxycholate or digitonin at 100 pg/mg protein, rather than by sonication. omission of the heating at 70°C of the sonicated and sodium dodecyl sulfate lysed sample, before electrophoresis, or performing the heating step at 37°C for four hours. had no obvious effect, on the resolution and proportion of thr discrete components identified here. It has been reported that the solubilization of yeast mitochondrial membranes in sodium dodecyl sulfate at alkaline pH causes the ronversion of a product of mibochondrial protein synthesis with an apparent molecular weight of 45,000 to a form with a molecular weight of about# 7800 (Tzagoloff $ Akai, 1972). No such phenomenon WAS detected in HeLa cells. Jn the electrophoretic runs shown in Figure 3 the samples had been dissolved in 2.5% sodium dodecyl sulfate/504 mercaptoethanol containing 2 x IO-’ M-NaOH, and incubated at 70°C for 30 minutes before the electrophoresis. When equivalent samples were dissolved in 2*50/, sodium dodecyl sulfate/50/a mernaptoethanol without NaOH addition, heated at 70°C and run through gels under identical condit,ions. no substantial difference in t(ha electrophoretia profiles from "0

298

I’.

COSTANTJNO

AND

G.

ATTARDT

those obtained with the alkaline samples was observed after either long runs (set c.g. Fig. 3(b)), or short runs. The same lack of effect of treatment with alkali or organic solvents, prior to electrophoresis, on the apparent molecular weight distribution of the mitochondrially synthesized polypeptides has been reported fo1 yea,st (Ebner et al., 1973). Tn an experiment utilizing a preparation labeled to a high specific activity with j3H]isoleucinc, material from the peak fraction of each one of components 1 to 8 isolated in a long electrophoretic run (see insert of Fig. 3(f)) was rerun through a. polyacrylamide gel. Components 2, 3, 4, 5 and 8 migrated as very sharp peaks, whereas somewhat broader peak.. were observed for components 1, 6 and 7 (Fig. 3(e) and (f)). Tn no case was there evidence of interconversion of t,hese components; however, each of them exhibited a certain amount of faster and slower moving maberial spread fairly uniformly throughout1 the gel, which presuma,bly derived from aggregation or disaggregation of thcb hetrrogenrous background underlying t)hr disc&e peaks in the original run.

TABLE

Mol~aular

wei@ts

1

of the products protein synthesis

(lomponrnt 1 2 3 4 6 0 7 8 9 10

Mol.

of mitochondrial

wt

42,000 39,000 36,000 31,500 27,600 24,000 22,500 19,600 lG,OOO 11,cioo

The values given above represent the averages of many estimtttes (5 or more). Each estimate was made on the basis of the electrophoretio mobility of the individual components relative to that of standard proteins run in a parallel gel. In the construct,ion of the individual standard curves, the apparent molecular weight of hnvine serum albumin (see Fig. 2(f) and legend) was used.

Table 1 shows the molecular weights of the discrete electrophoretic components described above, as estimated, in many experiments, from their mobility relative to that of standard proteins run in parallel gels. (b) Xolubilit2/

properties

of the mitochondrial

products

in chloroform/methanol

(i) Neutral chloroformlmethalaol extractiola The products of mitochondrial protein synthesis have been shown to be soluble to a variable extent in a mixture of chloroform and methanol (Kadenbach, 1971a; & Hadvbry, 1973: Tzagoloff & Akai, 1972 : Murray & Linnane, 197,)9. Kadenbach

MlTOCHUNlIi~IAL

PBOTEIN

SYKTHESLS

L”30

Burke & Beattie, 1973), a property described previously for brain proteolipid (Polch & Lees, 1951). This property was investigated in the HeLa cell system, using the mitochondrial fraction from cells that had been labeled for one hour with [“HIisoleucine in the presence of emetine. In order to compare the solubility properties of the products of mitochondrial and cytoplasmic protein synthesis that are present in the 5000 g,, mitochondrial fraction, the cells had been labeled for 48 hours with [14C]arginine and [l*C]lysine, two amino acids that show an extremely low apparent incorporation into the products of mitochondrial protein synthesis (Costantino & Attardi, 1973). A sample of this material was extracted at 50°C for 30 minutes with ;L 2: 1 neutral mixture of chloroform and methanol, as dcscribcd in Materials and Methods, section (e). About 28% of the initial 3H radioactivity \vas recovered in the organic phase, while less than l’l/o of the 14C-labeled material could be extra&cd under these conditions (Table 2). This represent’s a more than 30-fold purification of the products of mitochondrial protein synthesis. The results of another sirnilul extraction oii the same material arc prescntcd in Table 2. TABLE 2 Solubilizdiola

of poteias

of the mitochordrial

U>mpononts

of tho sonicated mitochondrial fraction

fraction

28 20

at 3f

lUG,UUUy,, pellet

3rJJJu0 gav pelloL

111~cl~lo~of~ont~ltt~ethut~ol

l’ercontage of cts/min solubilized Neutral extraction Acidic oxtractiou 14c 3H 1% 3H

1t Total

fmction

l!J

;: 39: 1t 2t

1

1st eslraction 2nd extraction 3rd extraction

10

2’3 I I 19

Cl -. -1

GO

20

40

11

36

11)

1 I

1 1 2 1

Samples containing 7000 to 37,000 “H cts/min and 6500 to 300,000 14C: cts/min (with a ratio of l*C to 3H varying between 0.6 and 8.0) were extracted with neutral or acidic chloroform/methanol mixtures at different temperatures and with/without methanol pretreatment, as specified below. t 5O”C, 30 min, 1 7092, 45 min, 3 GOT,!, 30 min,

no methanol. plus methanol. plus methanol.

Neutral chloroform/nwthanol extraction was also performed on the componeut~n of the sonicated mitochondrial fraction sedimenting at 30,000 g,, or 105,000 gay (Materials and Methods, section (d)). These two submitochondrial fractions contain products of mitochondrial protein synthesis that exhibit an electrophoretic profile comparable to that of the products extracted from the total mitochondrial fraction. With both fractions results similar to those described above were obtained; in all cases the purification achieved was about 20-fold ( see Table 2). It should be noted

300

I’.

cos’I’.~sTlsu

*IA-U

G.

Xl”l’dKLll

that repeated treatments with neutral chloroform/methanol did not increase the solubility of the mitochondrial products, since almost all the extractable radioactivity was recovered in the chloroform/methanol phase of the first extraction. Figure 4 shows the electrophoretic profiles of the proteins not extractable (a) and those extractable (b) by neutral chloroform/methanol from the total mitochondrial fraction, under the conditions described above (expt 2 in Table 2). Prom the comparison of the 3H radioactivity profiles in Figure (a) and (b) it appears that the extraction by neutral chloroform/methanol of the products of mitochondrial protein

3

h’rc. 4. Eleotrophoretio profiles of the proteins of the mitoohondrial fraction extraoted with neutral ohloroform/methanol and of the insoluble residue from HeLa cells long-term labeled with [i4C]arginine and [14C]lysine and pulse-labeled with [3H]iaoleuoine in the presence of emetine. HeLa cells were labeled for 48 h with [Wlarginine and [‘%]lysine (0.0063 @i/ml of each), and then for 1 h with [3H]isoleuoine (2.6 &i/ml) in the presence of emetine, as desoribed in Met&ah and Methods, section (b). A semple containing 0.5 mg protein of the mitoohondrisl fraction was extracted with 2 ml of a 2: 1 (v:v) mixture of chloroform and methanol (see Materials and Methods, Equivalent samples of the extract and of the insoluble residue section (e) and text for details). were eleotrophoresed for 30 h. (a) Insoluble residue. (b) Chloroform/methanol soluble material. --a-a---, [8H]isoleuoine-labeled proteins; --- 0-O--, l’%]arginine, [‘W]lysine-labelad proteins.

I\lITOC’HONL)l~lAL

P’tlO’l’ElS

SYS’I’HESIS

:Ml1

synthesis is to a certain degree selective. At least six of the discrete electrophoretic components with a molecular weight greater than 20,000 (components 2 to 7) are soluble to a variable extent in the organic mixture under the conditions described above of extraction; maximum solubility is exhibited by component 3. The most remarkable feature is the absence, in the chloroform/methanol extract, of peak 8, which is one of the major products of mitochondrial protein synthesis. However. it is possible to solubilizo partially the protein of peak 8 in neutral chloroform/ methanol by carrying out the extraction at 70°C for 45 minutes and pretreating the material with 909/, methanol at room temperature. Under these conditions, however. the overall efficiency of the organic extraction is substantislly reduced. III Figure 4(a) it appears that there is no obvious correspondence betwetln t,hc ‘jH radioactivity peaks, represnnt,ing the product(s of mit,ochondrial prot’ein synt’hwis. and t’hc profile of [‘*Cjarginint~- and ~14(!~lysine-lnbel~tl t’otal proteins of the mitechondrial fraction, indica,ting that, most of. if not a,11t’hese prokins arc synthesizal on q%oplasmic ribosomes. The very small amount of 14C radioactivity ~xtractahlc with neutral chloroform/met~hanol is spread t,hroughout the Irngt’h of the gel. (ii)

ilciu!ic

chhofor7i+rbe.th7bol

extracfio,b

A mitochondrial preparation from cells double labeled with [‘*C]argininc-[‘“C Ilysino and [3H]isoleucine, as described above, was subjected to extraction with an acidified chloroform/methanol mixture. For this purpose, the sonicated mitochondrial fraction was first treated at room temperature with 90:/A methanol, which did not solubilize any appreciable amount of protein, and then extracted at 70°C for 45 minutes with a 2 : 1 mixture of chloroform and methanol containing 2 x 10m3 M-HCl. About 40:/, of the [3H]isoleucine-labeled products of mitochondrial protein synthesis and a non-negligible fraction (about ll’$i) of the [14C]argininc- and [l*C llysinclabeled proteins were found in the organic phase. The quantitative results of the acidic chloroform/methanol extraction in this and other experiments carried out undrl different conditions are summarized in Table 2. It appears that in all oases,undcl acidic conditions, the chloroform/methanol mixture was more effective in solubilizing the proteins of the crude mitochondrial fraction, than at neutral pH. However, at acid pH, the selectivity of extraction of the proteins synthesized in mitochondria was to a great extent lost. The electrophoretic profiles of the proteins of the untreated doubly labeled mit,oohondrial fraction and of the proteins extract’ed with acidic chloroform/met,hanoi mixture in the experiment described above show that essentially all the productIs of mitochondrial protein synthesis are soluble to a similar extent in acidic chloroform/ methanol. On the other hand, of the cytoplasmic proteins present in the mitochondrial fraction, the components with lower molecular weight are preferentially extractable by t’he orga,nic mixture at acid pH.

4. Discussion In the present investigations, a progressive decreasein the overall rate of labeling of the mitochondrial protein products was observed in the presence of emetine, presumably reflecting an indirect effect of this drug on the rate of mitochondrial protein synthesis. Similar observations were previously made by using oycloheximide, another inhibitor of cytoplasmic protein synthesis, in different mitochondrial systems

302

P.

COSTANTlNO

RNL,

C:. A’L”‘l’i\RUI

(Sebald et OZ., 1969,1971). Several lines of evidence suggest that the indirect effect of inhibition of cytoplasmic protein synthesis on mitochondrial protein synthesis results from depletion of pools of cytoplasmically made proteins, with which the mitochondrial products must combine in order for their synthesis to continue (see Schatz & Mason, 1974). In the present work, the finding that all the identified discrete products of mitochondrial protein synthesis were already recognizable after a ten-minute: [3H]isoleucine pulse, i.e. at a time when the incorporation of the isotope was &ill fairly linear, suggests that the observed electrophorctic pattern qualitatively reflects the physiological situation. (a) Resolution of discrete electrophorekic corapouents awwtbg the products mitochondrial protein synthesis in HeLa cells

qf

In the present work, under the described conditions of sample preparation and gel electrophoresis, ten discrete components have been reproducibly observed, over a heterogeneous background, in the electrophoretic profiles of the newly synthesized products of mitochondrial protein synthesis labeled %nvivo with [3H]isoleucinc in the presence of emetine. It should be emphasized that it is not yet known whether each one of these electrophoretic components represents an individual polypeptide species, or whether more than one species is present in some of the peaks. Control experiments, involving a direct sodium dodecyl sulfate lysis of whole pulse-labeled cells or the use of the antiprotease inhibitor (PMSP, see Materials and Methods, section (c)) during cell homogenization and fractionation, tend to exclude the possibility that the discrete electrophoretic components observed here have resulted from enzymatic degradation during extraction. The resolution and reproducibility achieved in the present work in the electrophoretic separation of the products of mitochondrial protein synthesis, makes it possible to use this separation with confidence as an analytical tool in the study of the metabolic properties and genetic control of these product,s. Concerning the chloramphenicol-sensitive, heterogeneous labeled material that underlies the discrete components, it seems likely from its kinetics of labeling that it consists of immature mitochondrial protein products, i.e. nascent chains or complete polypeptides that have not yet acquired their final configuration by secondary chemical modifications (like covalent linkage of fatty acid or sugar moieties) or non-covalent association with phospholipids. The results of preliminary pulse-chase experiments (unpublished observations) are in agreement with this interpretation. Each one of the electrophoretic components 1 to 8, when rerun through a polyacrylamide gel, migrated as a sharp to fairly sharp peak to essentially the same position in the gel as in the first run, thus excluding a reversible aggregation as a mechanism of their formation. Furthermore, neither by alkali treatment nor chloroform/methanol extraction (see below) was a conversion observed of any of the higher molecular weight components to smaller size components, as reported by Tzagoloff & Akai (1972) for yeast mitochondrial protein synthesis products. No substantial change was observed in the relative labeling of the various discrctc: electrophoretic components during the first hour of emetine treatment, apart from a reproducible decrease in the relative labeling of component 5 and, less clearly, of components 9 and 10. The latter two components appeared to be always labeled to a comparatively low extent, and were sometimes difficult to recognize aga,inst the heterogeneous background, especially after longer labeling times. The metabolic

MlTOCHONDRIAL

behavior of the electrophoretic investigated.

PROTEIN

SYNTHESIS

303

components identified in this work is at present being

(b) Size and nature of the products of mitoclwndrial protein synthesis in HeLa cells A considerable amount of evidence (see review by Benttie, 1971) indicates that the products of mitochondrial protein synthesis are hydrophobic proteins of the inner mitochondrial membrane. Although it is not yet possible to identify the discrete electrophoretic components detected here with individual polypeptide species, it is interesting to notice that both the number and the size range of these components agree reasonably well with the number and size of the defined molecular species which llavr been identified in yeast and Neurospora mitochondria as products of mitochondrial protein synthesis, i.e. three subunits of the cytochrome c osidase (Ross et al.. 1974; Sebald et al., 1974; Rubin & Tzagoloff, 1973a,b; Mahler et al., 1974), four subunits of the oligomycin-sensitive ATPase (Tzagoloff & Meagher, 1972), one or two subunits of cytochrome b (Weiss & Ziganke, 1974), and one subunit of cytochrome c1 (Ross et al,, 1974). This correspondence is consistent with the evidence suggesting that, in animal cells, the products of mitochondrial protein synthesis are component,s of the same enzymatic complexes of the inner mitochondrial membrane as in lower eukaryotes (Kroon & DeVries, 1971; Kadenbach, 1971b; Storrie & Attardi, 1972,1973; see also review by Borst, 1972). Tzagoloff and collaborators have reported that the main product of mitochondrial protein synthesis in yeast is a small polypeptide with a molecular weight of 8000 to 9000, which is a subunit (subunit 9) of the oligomycin-sensitive ATPase complex (Tzagoloff & Akai, 1972; Sierra & Tzagoloff, 1973). Furthermore, they have observed that, in a neutral sodium dodecyl sulfate lysate of the mitochondrial membranes, this protein occurs most in a polymeric form, with an apparent molecular weight of 45,000. Conversion of the polymeric to the low molecular weight form of this protein WLLS achieved by treatment of the mitochondrial membranes with chloroform/methanol or by depolymerization in sodium dodecyl sulfate at alkaline pH. In the present work, no protein component of molecular weight 8000 to 9000 was observed in the electrophoretic profiles of sodium dodecyl sulfate lysates, either neutral or alkaline, of the sonicated mitochondrial fraction, or of chloroform/methanol extracts of the same fraction. Similarly, no small protein extractable with chloroform/methanol has been detectecl by Burke & Beattie (1973) among the proteins synthesized by isolated rat liver mitochondria. It is possible that, in animal cells, the conversion of the high to the low molecular weight form of this protein by treatment with base or organic solvent, under the same conditions applied successfully to yeast material, does not occur. The 11,500 molecular weight electrophoretic component (component lo), which was detected here after short labeling times, may represent the newly synthesized, low molecular weight form of the protein in question, equivalent to the 8000 to 9000 molecular weight polypeptide in yeast. It should be noted in this connection that evidence has been recently presented (Cattell et al., 1971; Stekhoven et al., 1972) for the occurrence in beef heart mitochondria of a small protein (10,000 to 13,000 molecular weight), which is soluble in chloroform/methanol and which binds specifically the inhibitor of oxidative phosphorylation, dicyclohexylcarbodiimide, as presumably does the subunit 9 of the ATPase complex in yeast (Sierra & Tzagoloff, 1973). Further invcst,igations are necessary to establish whether the elechrophoretic component 10

1’.

304

COSTANTINO

dND

C:. ATTARDI

detected in the present work, or some other as yet unidentified component,, is the equivalent of subunit 9 of the ATPase complex in yeast. It is interesting to mention that the number and size range of the discrete electrophoretic components identified here among the products of mitochondrial protein synthesis correlate in general fairly well also with the number and size of the p+(A)containing RNA components coded for by mitochonclrial DNA that have been recent]) described in HeLa cells (Ojala & Attardi, 1974). This correspondence suggests t’hat the latter components may be the templates for t’he synthesis of at least, some of the mit,ochondrially synthesized proteins. (c) &%&tbbility

in chloroformlmeth~anol

of the produch

of mitocho?adrid

p&in

synthesis

In the present work, 20 to 30% of the mitochondrially synthesized proteins labeled during a one-hour [3H]isoleucine pulse in the presence of emetine. and only I”/0 or less of the total proteins of the mitochondrial fraction (representing mostly cytoplasmically synthesized proteins) were solubilizecl with neutral chloroform/methanol : this represents a 20 to 30-fold purification of the mitochonclrial protein products, much higher than previously reported for rat liver material (Burke $ Beattie, 1973). The efficiency of extraction varied considerably between the different electrophoretic components, but at least six components with a molecular weight greater than 20,000 were extractable by neutral chloroform/methanol t’o an appreciable extent. The reason for the incompleteness of extraction of the various electrophoretic components by chloroform/methanol is unclear; it is not known what the physico-chemical basis of the solubility behavior of these proteins is, nor to what extent it can be influenced by changes occurring under the rather drastic conditions of extraction. As mentioned above, no conversion of a high molecular weight component to a protein smaller than 10,000, as reported for the yeast mitochonclrial product by Tzagoloff & Akai (1972). was observed. Nor was a 2000 molecular weight polypeptide detected in the chloroform/methanol extract, as described by Kadenbach (1971a). Our results are, on thta contrary. in gross agreement with those reportted by Burke & Beattie (1973) for thn products of protein synthesis by isolated rat liver mitochontlria, as concerns the preferential extraction by neutral chloroform/methanol of the higher molecula,r weight proteins. However, the limited resohltion of the electrophoretic analyses reportSedby the authors quo’ted ahove prevents a clet’ailetdcomparison of t,heir rrsults with the present ones. Chloroform/methanol extraction under acidic conditions proved to hc morr ef&:ient, hut less selective for the mitochondrially synthesized proteins, than the neutral extraction. in agreement with the results reported by Tzagoloff & Akai (1972). The selective solubility in organic solvents of the products of mitochondrial protein synthesis will undoubtedly facilitate the isolation in pure form of individual polypept,idessynthesized in the organelles, and the analysis of the contribution of mitoc:hondrial products to the formation of enzyme complexes of tJheinner mitochonclrial membrane. Theso inve&igations have been supported by a grant from t,he U.S. Public HcaltlL Service GM- 1 I7 26 rendby the Ente Nazionale Idrocarburi, Italy. l’ho able assistanceof MRArger Drew, MS Gloria Engel and MS RossellaUmmarino is gra.tnflllly acknowledged,

MITOCHONDRIAL

PROTEIN

SYNTHESIS

305

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